Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: Correlation to function in a growth cone collapse assay

Pabbisetty, Kumar; Yue, Xin; Li, Chen; Himanen, Juha P.; Zhou, Ran; Nikolov, Dimitar B.; Hu, Longqin

doi:10.1110/ps.062608807

Cited by 45 publications

(44 citation statements)

References 22 publications

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“…Avidity effects are strongly dependent on the valency of the ligand, as observed, for example, with binding of dimeric ephrin-A5 ligand to dimerized EphA3 receptor (45). Accordingly, the measured K d values for FKs to our CD46ex-Fc were about 5 logs lower for the Ad3/7-FKs and 3 logs lower for Ad11/35-FKs compared to the K d values for the monovalent CD46-SCR I-II interactions published by others (10,47,48,71,72).…”

Section: Discussionsupporting

confidence: 46%

Avidity Binding of Human Adenovirus Serotypes 3 and 7 to the Membrane Cofactor CD46 Triggers Infection

Trinh

Lesage

Chennamparampil

et al. 2012

J Virol

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The species B human adenoviruses (HAdVs) infect cells upon attaching to CD46 or desmoglein 2 (DSG-2) by one or several of their 12 fiber knob trimers (FKs). To test whether DSG-2 and CD46 simultaneously serve as virus receptors for adenovirus type 3 (Ad3), we performed individual and combined CD46/DSG-2 loss-of-function studies in human lung A549 and 16HBE14o cells. Our results suggest that in these cells, DSG-2 functions as a major attachment receptor for Ad3, whereas CD46 exerts a minor contribution to virus attachment and uptake in the range of ϳ10%. However, in other cells the role of CD46 may be more pronounced depending on, e.g., the expression levels of the receptors. To test if avidity allows Ad3/7 to use CD46 as a receptor, we performed gain-of-function studies. The cell surface levels of ectopically expressed CD46 in CHO or human M010119 melanoma cells lacking DSG-2 positively correlated with Ad3/7 infections, while Ad11/35 infections depended on CD46 but less on CD46 levels. Antibody-cross-linked soluble CD46 blocked Ad3/7/11/35 infections, while soluble CD46 alone blocked Ad11/35 but not Ad3/7. Soluble Ad3/7-FKs poorly inhibited Ad3/7 infection of CHO-CD46 cells, illustrating that Ad3/7-FKs bind with low affinity to CD46. This was confirmed by Biacore studies. Ad3/7-FK binding to immobilized CD46 at low density was not detected, unlike that of Ad11/35-FK. At higher CD46 densities, however, Ad3/7-FK bound to CD46 with only 15-fold-higher dissociation constants than those of Ad11/35-FK. These data show that an avidity mechanism for Ad3/7 binding to CD46 leads to infection of CD46-positive cells.

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Section: Discussionsupporting

confidence: 46%

Avidity Binding of Human Adenovirus Serotypes 3 and 7 to the Membrane Cofactor CD46 Triggers Infection

Trinh

Lesage

Chennamparampil

et al. 2012

J Virol

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“…Recently our studies have demonstrated that fluorescently labeled TM fragments of the EphA1 receptor self-associate in liposomes, forming helical dimers crossing the lipid bilayer (19). Consistent with this notion and with the fact that Eph-ephrin signaling requires lateral dimerization or aggregation in cell membrane (2,6,12,20), we present the high resolution spatial structure of the homodimeric TM domain of the human EphA1 receptor obtained by heteronuclear NMR in lipid bicelles combined with molecular dynamics (MD) relaxation in explicit membrane. The found dimerization specificity and dependence of structural-dynamic properties of the EphA1 TM domain on ionization state of the membrane embedded glutamate carboxyl group tempted us to speculate how extracellular conditions and local cell membrane environment can modulate biological activity of the receptor, providing a better understanding of the molecular mechanism of the Eph-ephrin signaling.…”

supporting

confidence: 55%

Spatial Structure and pH-dependent Conformational Diversity of Dimeric Transmembrane Domain of the Receptor Tyrosine Kinase EphA1

Bocharov

Mayzel

Volynsky

et al. 2008

Journal of Biological Chemistry

103

149

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Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel ␣-helical bundle, region (544 -569) 2 , through the N-terminal glycine zipper motif A 550 X 3 G 554 X 3 G 558 . Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu 547 , whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A 560 X 3 G 564 . This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK a value of Glu 547 and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.Erythropoietin-producing hepatocellular (Eph) 3 receptor and corresponding membrane-bound Eph receptor-interacting proteins (ephrins) transduce signal in a cell-cell contact-dependent fashion, thereby coordinating growth, differentiation, and patterning of almost every organ and tissue during vertebrate and invertebrate embryogenesis (1, 2). In adult organism, Eph-ephrin interactions can also trigger a wide array of cellular responses, including cell boundary formation, motility, adhesion, and repulsion, especially for neuronal and endothelial cells, whereas deregulated reemergence of Eph function appears to contribute to mechanism of tissue injury and of tumor invasion and metastasis. Intriguingly the Eph-ephrin interactions may have a role in synaptic plasticity, learning, memory formation, and mental disease (3, 4). The Eph receptors represent the largest family of receptor tyrosine kinases and are divided into subclasses A and B based on the sequence homology of their extracellular parts, the structure, and the binding affinity (5). Ephrin-A ligands share a membrane-tethered glycosylphosphatidylinositol anchor, whereas ephrin-B ligands have a transmembrane domain and a short cytoplasmic tail. The Eph receptors and ephrins are not only numerous, but their relationship is also complex (6). Receptor-liga...

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“…It should be noted that Fc fusion proteins are dimeric, which increases their binding avidity. 23,24 Nevertheless, the apparent K D values obtained with this method can be used to compare the binding strengths of different Eph receptor-ephrin combinations.…”

Section: Resultsmentioning

confidence: 99%

Profiling Eph receptor expression in cells and tissues

Noberini

Torre

Pasquale

2012

Cell Adhesion & Migration

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The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.

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Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: Correlation to function in a growth cone collapse assay

Cited by 45 publications

References 22 publications

Avidity Binding of Human Adenovirus Serotypes 3 and 7 to the Membrane Cofactor CD46 Triggers Infection

Avidity Binding of Human Adenovirus Serotypes 3 and 7 to the Membrane Cofactor CD46 Triggers Infection

Spatial Structure and pH-dependent Conformational Diversity of Dimeric Transmembrane Domain of the Receptor Tyrosine Kinase EphA1

Profiling Eph receptor expression in cells and tissues

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